Biochemical and Genetic Dissection of Chemokine Receptor CXCR3 Signaling

趋化因子是趋化细胞因子的超家族，通过激活G蛋白偶联受体来控制白细胞的运动。这些受体的信号结构域，以及它们相互作用的信号分子，还没有被阐明，这也是我们在之前的资助时期研究的重点。我们重点研究了趋化因子受体CXCR3及其配体CXCL10、CXCL9和CXCL11作为趋化因子诱导的信号转导模型，并探讨了哪些CXCR3结构域在体外参与了趋化和受体内化两种趋化因子诱导的功能。CXCR3在效应器T细胞上表达，在体内介导这些细胞的运输。趋化因子受体通过促进一过性细胞迁移诱导定向迁移 位于细胞前沿的活性信号复合体的定位。因此，细胞通过在空间上调节信号转导通路的活性来感知方向。在之前的资助期，我们启动了一些研究，以检验趋化是由一种称为“趋化复合体”的支架复合体介导的，该复合体通过与趋化因子受体本身的相互作用，将相关的下游介质保持在与激活受体密切相关的位置。我们已经发现CXCR3的C-Tail是诱导趋化所必需的，并在C-Tail中发现了两个重要的结构域。一个域 含有一个PDZ结合域，我们已经发现这是CXCRS在体外诱导趋化所必需的。此外，我们还发现一个候选的PDZ结构域支架蛋白GIPC与这个CXCR3 PDZ结合域相互作用，并且在CXCRS诱导的趋化作用中也是重要的。在目前的建议中，我们将确定CXCR3 PDZ结合域和PDZ支架蛋白GIPC在CXCR3介导的T细胞体内募集中的意义。在受体内化方面，我们发现CXCL11是CXCR3内化的生理诱导者，并且发现CXCR3的不同结构域介导CXCL11与CXCL9/10的CXCR3内化。这增加了CXCR3差异的可能性 内化是这3种CXCR3配体的一些不同生物学功能的基础。然而，受体内化在趋化因子引导的白细胞体内转运中的作用还不是很清楚。在目前的方案中，我们将通过研究CXCR3结构域在体外内化中的重要作用来确定受体内化在CXCRS诱导的T细胞体内转运中的作用。除了研究CXCR3信号域在体内T细胞转运中的作用外，我们还将利用RNAi技术和质谱学继续我们的研究，以确定CXCR3信号转导途径的新成分。 调节趋化物质信号转导从而导致白细胞定向迁移的分子途径对健康和疾病具有重要意义。一旦确定，这一途径的调节可能允许新的方法来调节免疫和炎症性疾病。

Chemokines are a super-family of chemotactic cytokines that control the movement of leukocytes by activating G protein-coupled receptors. The signaling domains of these receptors, and the signaling molecules with which they interact, have yet to be elucidated, and have been the focus of our studies in the prior funding period. We have focused our studies on the chemokine receptor CXCR3 and its ligands CXCL10, CXCL9, and CXCL11 as a model for chemokine-induced signal transduction, and have investigated which CXCR3 domains are involved in transducing two chemokine-induced functions in vitro, chemotaxis and receptor internalization. CXCR3 is expressed on effector T cells, where it mediates the trafficking of these cells in vivo. Chemokine receptors induce directed cell migration by promoting a transient localization of an active signaling complex at the leading edge of the cell. Cells thus sense direction by spatially regulating the activity of the signal transduction pathway. In the prior funding period, we initiated studies to test the hypothesis that chemotaxis is mediated by a scaffolding complex called the "chemotaxisome" that serves to hold relevant downstream mediators in close proximity to the activating receptor via interactions with the chemokine receptor itself. We have found that the C-tail of the CXCR3 is required for inducing chemotaxis and have identified two important domains within the C-tail. One domain contains a PDZ binding domain, which we have found is required for CXCRS-induced chemotaxis in vitro. In addition, we have found that a candidate PDZ domain scaffold protein, GIPC, interacts with this CXCR3 PDZ binding domain and is also important for CXCRS-induced chemotaxis. In the present proposal, we will determine the significance of the CXCR3 PDZ binding domain and the PDZ scaffold protein GIPC in CXCR3-mediated T cell recruitment in vivo. With regard to receptor internalization, we have found that CXCL11 is the physiological inducer of CXCR3 internalization, and have found that distinct domains of CXCR3 mediate CXCL11 vs. CXCL9/10 CXCR3 internalization. This raises the possibility that differences in CXCR3 internalization underlie some of the different biological functions of these 3 CXCR3 ligands. However, the role of receptor internalization in chemokine directed leukocyte trafficking in vivo is not well understood. In the present proposal, we will determine the role of receptor internalization in CXCRS-induced T cell trafficking in vivo by investigating the roles of the CXCR3 domains we have determined to be important for internalization in vitro. In addition to studies defining the roles of CXCR3 signaling domains in T cell trafficking in vivo, we will use RNAi technology and mass spectroscopy to continue our studies to define novel components of the CXCR3 signal transduction pathway. The molecular pathway that regulates chemoattractant signal transduction that results in the directed migration of leukocytes has important implications for health and disease. Once defined, the modulation of this pathway may allow for novel approaches to modulate immune and inflammatory diseases.